FIGURE 4.

LKB1 phosphorylates PAK1 in vitro at Thr¹⁰⁹. A, schematic presentation of PAK1 and its mutant forms. wt PAK1 contains the p21-binding domain (light gray) and the kinase domain (dark gray). The noncatalytic domains are in white. Thr¹⁰⁹ is a possible phosphorylation site for LKB1. B, LKB1 phosphorylates GST-PAK1-PBD at Thr¹⁰⁹ in vitro. The recombinant LKB1 complex (LKB1-STRAD-MO25) was incubated with GST-PBD or GST-PBD-T109A. C, LKB1 phosphorylates full-length GST-PAK1-K299R and GST-PAK1-T109A/K299R to a lesser extent. The in vitro kinase assays were performed using the recombinant LKB1 complex with GST-PAK1-K299R or GST-PAK1-T109A/K299R. The phosphorylation of the indicated recombinant proteins was visualized using BAS-5000 Bio-imaging Analyzer. These experiments were repeated at least twice with similar results.